Fluorescence lifetime imaging microscopy and time-resolved anisotropy of nanomaterial-induced changes to red blood cell membranes

With the use of engineered nano-materials (ENM) becoming more prevalent, it is essential to determine potential human health impacts. Specifically, the effects on biological lipid membranes will be important for determining molecular events that may contribute to both toxicity and suitable biomedical applications. To better understand the mechanisms of ENM-induced hemolysis and membrane permeability, fluorescence lifetime imaging microscopy (FLIM) was performed on human red blood cells (RBC) exposed to titanium dioxide ENM, zinc oxide ENM, or micron-sized crystalline silica. In the FLIM images, changes in the intensity-weighted fluorescence lifetime of the lipophilic fluorescence probe Di-4-ANEPPDHQ were used to identify localized changes to membrane. Time-resolved fluorescence anisotropy and FLIM of RBC treated with methyl-ss-cyclodextrin was performed to aid in interpreting how changes to membrane order influence changes in the fluorescence lifetime of the probe. Treatment of RBC with methyl-ss-cyclodextrin caused an increase in the wobble-in-a-cone angle and shorter fluorescence lifetimes of di-4-ANEPPDHQ. Treatment of RBC with titanium dioxide caused a significant increase in fluorescence lifetime compared to non-treated samples, indicating increased membrane order. Crystalline silica also increased the fluorescence lifetime compared to control levels. In contrast, zinc oxide decreased the fluorescence lifetime, representing decreased membrane order. However, treatment with soluble zinc sulfate resulted in no significant change in fluorescence lifetime, indicating that the decrease in order of the RBC membranes caused by zinc oxide ENM was not due to zinc ions formed during potential dissolution of the nanoparticles. These results give insight into mechanisms for how these three materials might disrupt RBC membranes and membranes of other cells. The results also provide evidence for a direct correlation between the size, interaction-available surface area of the nano-material and cell membrane disruption.